Retinal pigment epithelial (RPE) cells and photoreceptor (PR) cells are functionally and developmentally closely integrated. We are characterizing a 65 kD RPE-specific protein that is membrane-associated, hydrophobic, and dependent upon detergent solubilization for its extraction. In cell fractionation experiments this protein is found in the microsomal pellet. We have obtained its amino acid analysis and the sequences of several tryptic and chymotryptic peptides. Its amino terminus is blocked. We have cloned cDNA for this protein, which encodes a 533-residue protein unlike any others in the data base. The interphotoreceptor retinoid-binding protein (IRBP) is involved in the transport of retinoids, a functional relationship between the RPE and the PR. We have subcloned DNA fragments corresponding to integral repeats of bovine IRBP into a bacterial expression vector. The resultant expressed protein fragments were tested for their retinoid- binding functions. One of the fragments, which comprises the central two repeats, bound retinol. This finding indicates that all four repeats of IRBP are not required for retinoid-binding activity. The binding appears to be localized to the central region of the IRBP protein.